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• W2169959679 abstract "A proteomic analysis of the secretome of cultured dermal fibroblasts from patients with systemic sclerosis (SSc) and nephrogenic systemic fibrosis (NSF) was performed to identify proteins that reflect the fibrotic process. Confluent culture supernatants from three cell strains each of normal, SSc, and NSF dermal fibroblasts were pooled separately, and each pool was labeled with a specific fluorochrome. The three pools were electrophoresed together on two-dimension SDS gels, and protein differential expression was evaluated by quantitative fluorescence analysis. The secretome analysis identified 1694 spots per sample, among which 890 spots (52%) were differentially increased or decreased (more than twofold) in SSc fibroblasts, and 985 spots (58%) were differentially increased or decreased in NSF fibroblasts compared with normal fibroblasts. Mass spectrometry analysis was then used to identify the proteins that had increased by the greatest extent in both NSF and SSc secretomes. Three reticulocalbin family members were among the 10 most up-regulated proteins. Confocal microscopy results validated the differential increase of reticulocalbin-1 in affected SSc and NSF skin, and Western blot findings demonstrated its presence in SSc sera. The secretomes of both SSc and NSF fibroblasts display a pattern of shared changes compared with the normal fibroblast secretome. The differentially increased proteins reflect an activated fibroblast phenotype and may represent a specific “fibrosis signature” that can be used as a biomarker for fibrotic diseases. A proteomic analysis of the secretome of cultured dermal fibroblasts from patients with systemic sclerosis (SSc) and nephrogenic systemic fibrosis (NSF) was performed to identify proteins that reflect the fibrotic process. Confluent culture supernatants from three cell strains each of normal, SSc, and NSF dermal fibroblasts were pooled separately, and each pool was labeled with a specific fluorochrome. The three pools were electrophoresed together on two-dimension SDS gels, and protein differential expression was evaluated by quantitative fluorescence analysis. The secretome analysis identified 1694 spots per sample, among which 890 spots (52%) were differentially increased or decreased (more than twofold) in SSc fibroblasts, and 985 spots (58%) were differentially increased or decreased in NSF fibroblasts compared with normal fibroblasts. Mass spectrometry analysis was then used to identify the proteins that had increased by the greatest extent in both NSF and SSc secretomes. Three reticulocalbin family members were among the 10 most up-regulated proteins. Confocal microscopy results validated the differential increase of reticulocalbin-1 in affected SSc and NSF skin, and Western blot findings demonstrated its presence in SSc sera. The secretomes of both SSc and NSF fibroblasts display a pattern of shared changes compared with the normal fibroblast secretome. The differentially increased proteins reflect an activated fibroblast phenotype and may represent a specific “fibrosis signature” that can be used as a biomarker for fibrotic diseases. Fibrotic disorders, which include systemic sclerosis (SSc), idiopathic pulmonary fibrosis, cirrhosis of the liver, and the newly recognized nephrogenic systemic fibrosis (NSF), are characterized by abnormal and excessive deposition of collagen and other extracellular matrix components in various tissues.1Varga J Abraham D Systemic sclerosis: a prototypic multisystem fibrotic disorder.J Clin Invest. 2007; 117: 557-567Crossref PubMed Scopus (940) Google Scholar, 2Rosenbloom J Castro SV Jimenez SA Fibrotic diseases: cellular and molecular mechanisms and novel therapies (Physiology in Medicine Series).Ann Intern Med. 2010; 152: 159-167Crossref PubMed Google Scholar, 3Gharaee-Kermani M Phan SH Molecular mechanisms of and possible treatment strategies for idiopathic pulmonary fibrosis.Curr Pharm Des. 2005; 11: 3943-3971Crossref PubMed Scopus (65) Google Scholar, 4Tsukada S Parson CJ Rieppe RA Mechanisms of liver fibrosis.Clin Chim Acta. 2006; 364: 33-60Crossref PubMed Scopus (333) Google Scholar, 5Cowper SE Su LD Bhawan J Robin HS LeBoit PE Nephrogenic fibrosing dermopathy.Am J Dermatopathol. 2001; 23: 383-393Crossref PubMed Scopus (426) Google Scholar Although their etiologies are probably quite diverse, the presence of extracellular matrix-producing fibroblasts displaying an activated phenotype in the affected tissues is typical of all fibrotic diseases. Fibroblast activation is characterized by a marked increase in the transcriptional activity of the genes encoding type I and type III collagens and fibronectin, initiation of the expression of α-smooth muscle actin, and the reduction of extracellular matrix degradative activities.6LeRoy EC Connective tissue synthesis by scleroderma skin fibroblasts in cell culture.J Exp Med. 1972; 135: 1351-1362Crossref PubMed Scopus (162) Google Scholar, 7Jelaska A Arakawa M Broketa G Korn JH Heterogeneity of collagen synthesis in normal and systemic sclerosis skin fibroblasts. Increased proportion of high collagen-producing cells in systemic sclerosis fibroblasts.Arthritis Rheum. 1996; 39: 1338-1346Crossref PubMed Scopus (103) Google Scholar, 8Jiménez SA Feldman G Bashey RI Bienkowski R Rosenbloom J Co-ordinate increase in the expression of type I and type III collagen genes in progressive systemic sclerosis fibroblasts.Biochem J. 1986; 237: 837-843Crossref PubMed Scopus (158) Google Scholar, 9Jiménez SA Saitta B Alterations in the regulation of expression of the α 1(I) collagen gene (COL1A1) in systemic sclerosis (scleroderma).Springer Semin Immunopathol. 1999; 21: 397-414PubMed Google Scholar Activated fibroblasts display contractile properties resulting from the expression of stress fibers containing α-smooth muscle actin, and their profibrotic activation is part of a complex set of molecular and biochemical changes that are conserved for multiple sequential passages in vitro.6LeRoy EC Connective tissue synthesis by scleroderma skin fibroblasts in cell culture.J Exp Med. 1972; 135: 1351-1362Crossref PubMed Scopus (162) Google Scholar, 7Jelaska A Arakawa M Broketa G Korn JH Heterogeneity of collagen synthesis in normal and systemic sclerosis skin fibroblasts. Increased proportion of high collagen-producing cells in systemic sclerosis fibroblasts.Arthritis Rheum. 1996; 39: 1338-1346Crossref PubMed Scopus (103) Google Scholar, 10Kirk TZ Mark ME Chua CC Chua BH Mayes MD Myofibroblasts from scleroderma skin synthesize elevated levels of collagen and tissue inhibitor of metalloproteinase (TIMP-1) with two forms of TIMP-1.J Biol Chem. 1995; 270: 3423-3428Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar The most frequent systemic fibrotic disorder is SSc, a disease characterized by excessive deposition of collagen and other connective tissue macromolecules in skin and multiple internal organs, prominent and often severe alterations in the microvasculature, and humoral and cellular immunological abnormalities. The most apparent and almost universal clinical features of SSc are related to the severe fibrotic changes occurring in multiple tissues and very prominently in the microvasculature.1Varga J Abraham D Systemic sclerosis: a prototypic multisystem fibrotic disorder.J Clin Invest. 2007; 117: 557-567Crossref PubMed Scopus (940) Google Scholar, 2Rosenbloom J Castro SV Jimenez SA Fibrotic diseases: cellular and molecular mechanisms and novel therapies (Physiology in Medicine Series).Ann Intern Med. 2010; 152: 159-167Crossref PubMed Google Scholar The extent and rate of progression of tissue fibrosis is of paramount importance in determining the clinical features and the prognosis of SSc. Indeed, fibrosis of the skin correlates with both survival and functional limitations.11Steen VD Medsger Jr, TA Improvement in skin thickening in systemic sclerosis associated with improved survival.Arthritis Rheum. 2001; 44: 2828-2835Crossref PubMed Scopus (190) Google Scholar, 12Clements PJ Hurwitz EL Wong WK Siebold JR Mayes M White B Wigley F Weisman M Barr W Moreland L Medsger Jr, TA Steen VD Martin RW Collier D Weinstein A Lally E Varga J Weiner SR Andrews B Abeles M Furst DE Skin thickness score as a predictor and correlate of outcome in systemic sclerosis: high-dose versus low-dose penicillamine trial.Arthritis Rheum. 2000; 43: 2445-2454Crossref PubMed Scopus (246) Google Scholar, 13Seibold JR McCloskey DA Skin involvement as a relevant outcome measure in clinical trials of systemic sclerosis.Curr Opin Rheumatol. 1997; 9: 571-575Crossref PubMed Scopus (25) Google Scholar, 14Denton CP Black CM Abraham DJ Mechanisms and consequences of fibrosis in systemic sclerosis.Nat Clin Pract Rheumatol. 2006; 2: 134-144Crossref PubMed Scopus (156) Google Scholar Although there has been substantial interest in the identification of biomarkers that allow early diagnosis and assessment of disease activity or that carry a predictive prognostic in SSc15Doran JP Veale DJ Biomarkers in systemic sclerosis.Rheumatology (Oxford). 2008; 47: v36-v38Crossref PubMed Scopus (10) Google Scholar, 16Hummers LK Biomarkers of vascular disease in scleroderma.Rheumatology (Oxford). 2008; 47: v21-v22Crossref PubMed Scopus (7) Google Scholar, 17Castro SV Jimenez SA Biomarkers in systemic sclerosis.Biomarkers Med. 2010; 4: 133-147Crossref PubMed Scopus (57) Google Scholar including global gene expression and microarray studies,18Sargent JL Milano A Connolly MK Whitfield ML Scleroderma gene expression and pathway signatures.Curr Rheumatol Rep. 2008; 10: 205-211Crossref PubMed Scopus (13) Google Scholar, 19Farina G Lafyatis D Lemaire R Lafyatis R A four-gene biomarker predicts skin disease in patients with diffuse cutaneous systemic sclerosis.Arthritis Rheum. 2010; 62: 580-588Crossref PubMed Scopus (126) Google Scholar fully validated biomarkers reflecting the fibrotic process are not available. The clinical semiquantitative assessment of skin thickness by palpation (modified Rodnan skin score) is considered the gold standard and the only primary outcome measure used in clinical trials of SSc disease-modifying agents. This subjective and highly variable assessment is fraught with inaccuracies as pointed out recently.20Clements PJ Lachenbruch PA Seibold JR Zee B Steen VD Brennan P Silman AJ Allegar N Varga J Massa M Wigley FM Ingenito F Weisman M White B Martin RW McCloskey D Moreland LW Mayes M Lally EV Unanue M Collier DH Weiner S Weinstein A Medsger Jr, TA Andrews B Dixon M Furst DE Skin thickness score in systemic sclerosis: an assessment of interobserver variability in 3 independent studies.J Rheumatol. 1993; 20: 1892-1896PubMed Google Scholar It is, therefore, generally accepted that the development of objective and reliable markers reflecting the severity of tissue fibrosis would be of great value for improving the performance of clinical trials and the accurate assessment of the efficacy of a given treatment. Furthermore, such markers would allow a reduction in the number of patients needed for clinical trials to achieve statistical power and would offer an objective and quantitative method independent of the subjective assessment of the investigators involved in the study. NSF is a recently recognized fibrotic disorder occurring in patients with renal insufficiency after exposure to Gd-containing contrast agents used for magnetic resonance imaging.5Cowper SE Su LD Bhawan J Robin HS LeBoit PE Nephrogenic fibrosing dermopathy.Am J Dermatopathol. 2001; 23: 383-393Crossref PubMed Scopus (426) Google Scholar, 21Jiménez SA Artlett CM Sandorfi N Derk C Latinis K Sawaya H Haddad R Shanahan JC Dialysis-associated systemic fibrosis (nephrogenic fibrosing dermopathy): study of inflammatory cells and transforming growth factor beta1 expression in affected skin.Arthritis Rheum. 2004; 50: 2660-2666Crossref PubMed Scopus (197) Google Scholar, 22Mendoza FA Artlett CM Sandorfi N Latinis K Piera-Velazquez S Jiménez SA Description of 12 cases of nephrogenic fibrosing dermopathy and review of the literature.Semin Arthritis Rheum. 2006; 35: 238-249Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 23Grobner T Prischl FC Gadolinium and nephrogenic systemic fibrosis.Kidney Int. 2007; 72: 260-264Crossref PubMed Scopus (246) Google Scholar Affected tissues from patients with NSF display a remarkable fibrotic process and, like SSc fibroblasts, fibroblasts cultured from patients with NSF produce increased levels of collagens and other extracellular matrix proteins, which are maintained in vitro for several passages.24Piera-Velazquez S Louneva N Wermuth PJ Fertala J Del Galdo F Jimenez SA Persistent activation of dermal fibroblasts from patients with gadolinium-associated Nephrogenic Systemic Fibrosis.Ann Rheum Dis. 2010; https://doi.org/10.1136/ard.2009.127761Crossref PubMed Scopus (33) Google Scholar, 25Edward M Fitzgerald L Thind C Leman J Burden AD Cutaneous mucinosis associated with dermatomyositis and nephrogenic fibrosing dermopathy: fibroblast hyaluronan synthesis and the effect of patient serum.Br J Dermatol. 2007; 156: 473-479Crossref PubMed Scopus (37) Google Scholar Here, we describe the results of a proteomic analysis of the secretome of fibroblasts from patients with SSc and NSF, which allowed the identification of several shared proteins that were substantially elevated in comparison with the secretome of normal fibroblasts and, thus, they may reflect the increased fibrogenesis of these cells. These proteins should therefore be considered putative biomarkers that may be useful to assess the extent and severity of the fibrotic process in fibrotic diseases. Dermal fibroblasts were isolated from punch or excisional biopsy samples of affected forearms obtained from patients with SSc fulfilling the American Rheumatism Association criteria for disease classification26Subcommittee for Scleroderma Criteria of the American Rheumatism Association Preliminary criteria for the classification of systemic sclerosis (scleroderma).Arthritis Rheum. 1980; 23: 581-590Crossref PubMed Scopus (4935) Google Scholar and from patients with a typical clinical presentation of NSF associated with recent exposure to Gd-containing magnetic resonance imaging contrast agents described previously.21Jiménez SA Artlett CM Sandorfi N Derk C Latinis K Sawaya H Haddad R Shanahan JC Dialysis-associated systemic fibrosis (nephrogenic fibrosing dermopathy): study of inflammatory cells and transforming growth factor beta1 expression in affected skin.Arthritis Rheum. 2004; 50: 2660-2666Crossref PubMed Scopus (197) Google Scholar, 22Mendoza FA Artlett CM Sandorfi N Latinis K Piera-Velazquez S Jiménez SA Description of 12 cases of nephrogenic fibrosing dermopathy and review of the literature.Semin Arthritis Rheum. 2006; 35: 238-249Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar All patients with SSc had the diffuse cutaneous subset as defined by LeRoy et al,27LeRoy EC Black C Fleischmajer R Jablonska S Krieg T Medsger Jr, TA Rowell N Wollheim F Scleroderma (systemic sclerosis): classification, subsets and pathogenesis.J Rheumatol. 1988; 15: 202-205PubMed Google Scholar and in all patients the disease was of recent onset (<18 months) and rapidly progressive. The patients with SSc and NSF from whom the biopsy samples were obtained were matched for age and sex and had not received corticosteroids, antifibrotic therapy, or immunosuppressive therapy. Punch biopsy samples from age- and sex-matched normal subjects were used as controls. The biopsy samples, processed within 1 hour of excision, were split in two halves: one half was formalin-fixed and paraffin-embedded for histopathological and immunohistochemical analysis, and the other half was processed for establishment of dermal fibroblast cell strains. For this purpose, the skin biopsy samples were minced with a scalpel, and small pieces of tissue were placed on plastic culture dishes and then covered with tissue culture medium, which was changed every 3 to 5 days until visible outgrowth of cells was obtained in approximately 2 to 3 weeks. The cells were enzymatically disassociated with 1 mg/ml trypsin at 37°C for 5 to 30 minutes and then subcultured exactly as described by LeRoy.6LeRoy EC Connective tissue synthesis by scleroderma skin fibroblasts in cell culture.J Exp Med. 1972; 135: 1351-1362Crossref PubMed Scopus (162) Google Scholar These culture conditions allowed the expansion of pure fibroblast populations without any contamination with epithelial or endothelial cells or cells of hematopoietic origin. Dermal fibroblasts from patients with SSc or NSF were subcultured and used between passages 5 and 6 to avoid loss of the fibroblast overproducer phenotype, which is preserved for at least 12 serial passages under these in vitro culture conditions.6LeRoy EC Connective tissue synthesis by scleroderma skin fibroblasts in cell culture.J Exp Med. 1972; 135: 1351-1362Crossref PubMed Scopus (162) Google Scholar, 17Castro SV Jimenez SA Biomarkers in systemic sclerosis.Biomarkers Med. 2010; 4: 133-147Crossref PubMed Scopus (57) Google Scholar Normal fibroblasts were also matched for passage number. Cell strains were established from three different patients with SSc, three different patients with NSF, and three normal individuals. All cultures were grown to confluence in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with vitamins (Cellgro, Manassas, VA), 10% fetal bovine serum (Invitrogen), and antibiotics (Cellgro). Once the cultures reached confluence, they were washed twice and incubated for 18 hours in serum-free Dulbecco's modified Eagle's medium. Supernatants from the three SSc, three normal, or three NSF cultures were pooled separately, and each pool was concentrated 40-fold by 18 hours of centrifugation on Centri-Sep 3000-kDa (Applied Biosystems, Carlsbad, CA) columns at 4°C, according to the manufacturer's instructions. To avoid any differences in the starting sample, the same volume of tissue culture media (20 ml) obtained when the cells reached overconfluence for at least 24 hours was used in the final step. Protein concentration in the culture supernatants was determined using a Quant Kit (GE Healthcare, Piscataway, NJ). The samples were then brought to pH 8 to 8.5 with 1 mol/L NaOH to optimize fluorescent tagging. For each gel, 50 μg of protein for each category was added to 400 pmol/L concentrations of Cy2, Cy3, or Cy5 fluorescent tags and allowed to incubate on ice for 30 minutes. The labeling reaction was quenched by addition of 1 μl of 10 mmol/L lysine and subsequent incubation on ice for 15 minutes. For two-dimensional (2D) electrophoresis, the three samples (normal, SSc, and NSF) were pooled, brought up to 350 μl in 8 mol/L urea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and supplemented with dithiothreitol (final concentration 13 mmol/L) and IPG buffer 3-10 (final concentration 2%) before 2D electrophoresis as described previously.28Sharma K Lee S Han S Francos B McCue P Wassell R Shaw MA RamachandraRao SP Two-dimensional fluorescence difference gel electrophoresis analysis of the urine proteome in human diabetic nephropathy.Proteomics. 2005; 5: 2648-2655Crossref PubMed Scopus (117) Google Scholar, 29Ramachandra Rao SP Wassell R Shaw MA Sharma K Profiling of human mesangial cell subproteomes reveals a role for calmodulin in glucose uptake.Am J Physiol Renal Physiol. 2007; 292: F1182-F1189PubMed Google Scholar For each gel, 18-cm, pH 3 to 10 immobilized pH gradient (IPG) strips were rehydrated at 30 V for 12 hours in 350 μl of sample, using an IPGphor (GE Healthcare). Once rehydration was complete, samples were focused at 500 V for 1 hour, 1000 V for 1 hour, and finally 8000 V for 6 hours. Immediately after completion, IPG strips were processed for separation by SDS-polyacrylamide gel electrophoresis. To reduce the disulfide bonds in the focused proteins in preparation for the second dimension, IPG strips were incubated for 15 minutes in equilibration buffer I consisting of 0.375 mol/L Tris-HCl, pH 8.8, 6 mol/l urea, 2% SDS, 20% glycerol, and 13 mmol/L dithiothreitol. The IPG strips were soaked in equilibration buffer II for an additional 15 minutes to alkylate the sulfhydryl groups. Buffer II is identical to buffer I with the exception that 2.5% (w/v) iodoacetamide is used instead of dithiothreitol. The strips were embedded in 0.7% w/v agarose on top of 12.5% acrylamide slab gels. Second-dimension separations were performed on a DALT6 platform (GE Healthcare). IPG strips were electrophoresed at 2 W/gel for 30 minutes, followed by 20 W/gel until the dye front reached the bottom of the gel. The gel was rinsed in deionized water and scanned using the difference in gel electrophoresis (DIGE)-enabled Typhoon Scanner (GE Healthcare). After scanning of the images, DeCyder 5.01 software (GE Healthcare) was used for differential gel analysis. The 2D images from the different samples were then compared using the DIA module of DeCyder with a value of 1000 as the initial estimate of protein spots present. DIA analysis allows for the direct comparison of intensities of specific protein spots between different samples within the same gel.30Karp NA Kreil DP Lilley KS Determining a significant change in protein expression with DeCyder™ during a pair-wise comparison using two-dimensional difference gel electrophoresis.Proteomics. 2004; 4: 1421-1432Crossref PubMed Scopus (149) Google Scholar The 2D gel was poststained with SYPRO Ruby and rescanned using the Typhoon Scanner. The resulting gel images were matched back to the master DIGE image and spots of interest were designated for picking from the SYPRO image. Protein spots of interest were prepared for mass spectral analysis by the Spot Handling Workstation (GE Healthcare). SYPRO-stained spots of interest were automatically cut from the gel and washed twice with 50 mmol/L ammonium bicarbonate in 50% methanol. The plugs were then dehydrated in 75% acetonitrile for 10 minutes and dried under a stream of air. Trypsin (10 μl of 20 μg/ml in 20 mmol/L ammonium bicarbonate) was added to each plug and incubated for 2 hours at 37°C. After digestion, the resulting tryptic peptides were extracted twice into 50% acetonitrile, 0.1% formic acid and then dried completely under a stream of air. Proteins were identified via liquid chromatography-mass spectrometry using a Thermo Scientific ProteomeX Workstation consisting of a Surveyor high-performance liquid chromatograph front end, followed by an LCQ DecaXP Plus ion-trap mass spectrometer. The dried-down peptide extracts were resuspended in 15 μl of 1% formic acid, and 10 μl was loaded onto a Thermo Hypersil-Keystone BioBasic C18 column (0.18 × 100 mm). The peptides were separated at a flow rate of 4 μl/min using a linear gradient of 2 to 50% acetonitrile in 0.1% formic acid over 45 minutes. As the peptides were eluted from the column, they were subjected to a full mass spectrometry scan, followed by tandem mass spectrometry of the three largest peaks. The resulting tandem mass spectrometry spectra were analyzed using SEQUEST. Protein identifications were considered valid if they met the Human Proteome Organisation (HUPO) protein identification filter (Xcorr ≥ 1.9, 2.2, or 3.75 for z = 1, 2, and 3, respectively; ΔCN ≥ 0.1; and Rsp ≤ 4) as described previously in the Human Plasma Proteome Project.31Omenn GS States DJ Adamski M Blackwell TW Menon R Hermjakob H Apweiler R Haab BB Simpson RJ Eddes JS Kapp EA Moritz RL Chan DW Rai AJ Admon A Aebersold R Eng J Hancock WS Hefta SA Meyer H Paik YK Yoo JS Ping P Pounds J Adkins J Qian X Wang R Wasinger V Wu CY Zhao X Zeng R Archakov A Tsugita A Beer I Pandey A Pisano M Andrews P Tammen H Speicher DW Hanash SM Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database.Proteomics. 2005; 5: 3226-3245Crossref PubMed Scopus (687) Google Scholar To validate the results obtained by the proteomic analyses, reticulocalbin (RCN)-1, one of the proteins that displayed the highest differential expression, was selected for semiquantitative assessment of its abundance in normal, SSc, and NSF skin. RCN-1 was analyzed by immunofluorescence using a RCN-1 rabbit polyclonal antibody (Bethyl Laboratories, Montgomery, TX). Isotype control staining was performed using rabbit IgG (Sigma-Aldrich, St. Louis, MO). Secondary antibodies were affinity-purified sheep (FAb)2 anti-rabbit IgG-Cy3 conjugated (Sigma-Aldrich). Paraffin-embedded sections from skin were deparaffinized with two changes of xylene for 10 minutes each and then two changes of ethanol for 5 minutes each. Antigen retrieval was performed by boiling the tissue sections in 10 mmol/L citrate buffer, pH 6.0, for 20 minutes. The sections were then rinsed in PBS for 2 minutes and incubated with 5% normal sheep serum for 20 minutes at room temperature to block nonspecific binding sites. The primary antibody incubation step was performed overnight at 4°C followed by incubation with the sheep polyclonal antibody (1:200). The unbound antibodies were removed from the sections after each incubation with three changes of PBS for 2 minutes each. Tissue sections were counterstained with 4,6-diamidino-2-phenylindole and analyzed using a Zeiss LSM 510 META confocal laser scanning microscope system. Zeiss META confocal software was used in balancing signal strength. The breakthrough of the 4,6-diamidino-2-phenylindole signal into the red and the green channel was recorded separately and subtracted from the 4,6-diamidino-2-phenylindole blue channel. Zeiss META enhancement software was used in balancing the signal strength, and the image was scanned eightfold to separate signal from noise. Panels were assembled using Photoshop software without any RGB modification. Sera from 10 patients with SSc and two healthy normal subjects (volunteer laboratory personnel) were assayed for RCN-1. All SSc sera were from patients who fulfilled the American Rheumatism Association classification criteria for SSc26Subcommittee for Scleroderma Criteria of the American Rheumatism Association Preliminary criteria for the classification of systemic sclerosis (scleroderma).Arthritis Rheum. 1980; 23: 581-590Crossref PubMed Scopus (4935) Google Scholar and with the diffuse subset of the disease classified according to LeRoy et al.27LeRoy EC Black C Fleischmajer R Jablonska S Krieg T Medsger Jr, TA Rowell N Wollheim F Scleroderma (systemic sclerosis): classification, subsets and pathogenesis.J Rheumatol. 1988; 15: 202-205PubMed Google Scholar Sera were diluted 1:10 with saline solution and electrophoresed on a 10% Tris-glycine SDS gel. Primary antibody against human RCN-1 (Bethyl Laboratories) was used at a 1:1000 dilution, incubated overnight at 4°C. Anti-rabbit horseradish peroxidase-conjugated antibodies at 1:5000 dilution were used as secondary antibody. We analyzed by 2D DIGE the supernatants from three SSc, three NSF, and three normal fibroblast cultures. The secreted proteins from normal fibroblast cell strains were pooled and conjugated with Cy2 fluorochrome, the secreted proteins from the three SSc fibroblast cell strains were pooled and conjugated with Cy3, and the secreted proteins from the three NSF cell strains were pooled and conjugated with Cy5. The images detected for each fluorochrome are shown in Figure 1. A total of 1694 spots were detected on the 2D electrophoresis. DIGE software analysis allowed quantitative determination of each fluorescent signal in every detected spot. The differential analysis of SSc versus NSF fibroblast secretomes revealed that 1403 spots (85%) had similar levels, whereas 65 spots were increased in the SSc sample and 226 spots were increased in the NSF sample (Figure 2A). In contrast, the volume ratio analysis comparison of the secretome of normal fibroblasts with that of SSc fibroblasts indicated that 450 spots were increased (more than twofold) in the SSc fibroblast secretome, whereas 440 were decreased (<50%) and 804 had similar levels (Figure 2B). The differential analysis of NSF versus normal fibroblast secretome identified 391 spots increased in NSF, 590 decreased, and 713 with similar expression (Figure 2C).Figure 2Differential analysis of fluorescence intensity. A: Differential analysis of SSc versus NSF fibroblast secretomes, showing 1403 spots (85%) with similar levels with 65 spots increased (green) in the SSc sample and 226 spots increased in the NSF sample (red). B: Volume ratio analysis comparison of the secretomes of normal fibroblasts and SSc fibroblasts, showing 450 spots increased (more than twofold) in the SSc fibroblast secretome, 440 spots decreased (<50%), and 804 spots with similar levels. C: Differential analysis of NSF versus normal fibroblast secretomes showing 391 spots increased in NSF, 590 spots decreased, and 713 spots with similar levels.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Each protein spot fluorescence intensity was calculated by DIGE software and the ratios between SSc and normal and NSF and normal were calculated (Figure 3A). To analyze the differentially expressed proteins with common quantitative changes among the NSF and SSc fibroblast secretomes, each spot was assigned a progressive number, and the fluorescence intensity ratio between SSc fibroblast secretome and normal fibroblast secretome was plotted against the spot number. The same analysis was performed for the volume ratio between the NSF and normal fibroblast secretomes and the two series of dot plots were overlaid in the same grap" @default.
• W2169959679 created "2016-06-24" @default.
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• W2169959679 date "2010-10-01" @default.
• W2169959679 modified "2023-10-02" @default.
• W2169959679 title "Proteomic Analysis Identification of a Pattern of Shared Alterations in the Secretome of Dermal Fibroblasts from Systemic Sclerosis and Nephrogenic Systemic Fibrosis" @default.
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• W2169959679 doi "https://doi.org/10.2353/ajpath.2010.091095" @default.
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